An effective immune response against microbial agents must be of both appropriate magnitude and type. Type 1 cell-mediated immunity relies on differentiation of a subset of T helper lymphocytes (Th1 cells) that induces both inflammatory and cytotoxic responses essential for destruction of intracellular pathogens such as Mycobacterium tuberculosis and Francesella tularensis. Generation of humoral (Type 2) immunity requires development of the Th2 subset that signals B cells to produce antibodies to ward off viruses. Small molecules and biologics that can effectively and selectively modulate these two prototypic responses represent the next generation of therapeutic adjuvants to boost vaccine-based protection against microbial agents. We have discovered two gene products that represent early molecular checkpoints in the development of each of these immune response types. T-bet is a transcription factor that dictates the Th1 genetic program while the XBP- 1 transcription factor controls the generation of plasma cells and hence antibodies. Thus, intracellular expression of the T-bet protein results in the development of the Th1 subset and the efficient generation of Type 1 immunity, while expression of XBP-1 results in antibody production and Type 2 immunity. Proof-of-principle studies in mouse models of Mycobacterium tuberculosis and Francesella tularensis in collaboration with Dr. Kramnik (Project 3) will be accompanied by parallel studies in human patients with active M tuberculosis infection to search for functionally relevant T-bet polymorphisms. Since T-bet is expressed in CD8 cells and dendritic cells but does not control IFNgamma, production in the former, efforts will be made to understand its role in these two cell populations. We have early evidence that T-bet may modulate the generation of CD8 effector cells, key regulators of immunity to pathogens. The interaction of T-bet with other genetic loci (Kramnik, Project 3) and its relationship to the genetics of the pathogen itself (Rubin, Project 4) will be established. Enhancement of endogenous T-bet expression or activity in T cells or XBP-1 in B cells are attractive therapeutic strategies. One approach requires understanding the transcriptional regulation of the T-bet gene. A second approach which will be undertaken with AlleCure (subcontract) involves high through put screening efforts using available libraries of compounds to identify small molecules that boost T-bet and XBP-1 activity and should provide another means to increase endogenous activity This latter approach does not necessarily rely on a priori knowledge about the regulation of either of these genes. An additional approach to modulating the activity of these two transcription factors relies on obtaining their crystal structures (Petsko, Project 5). Structures of these potential drug targets will provide a framework for virtual screening of combinatorial libraries and for structure-guided drug design.